TB is an infection caused by species of the Mycobacterium tuberculosis complex (MTBC). In 2014, 1.5 million people died from the disease, which is unacceptable given that TB is curable. The lack of a technology for accurate TB detection in limited-resource settings is a major obstacle in the fight against the disease. However, available detection methods have poor performance and are not suited for diagnosis in limited resource settings. The most commonly used method to detect TB is smear microscopy, a procedure developed more than 100 years ago that only detects 50% of the cases. Here, we propose to develop a next generation molecular diagnostic test that can dramatically improve TB diagnosis. The new platform will be based on a novel technology capable of detecting single molecules using simple equipment. In the single molecule technique, probe hybridization to a target nucleic acid is directly detected without PCR amplification. The technique is capable of detecting 50 CFU/ml of Mycobacterium tuberculosis cells which is below the limit of detection of the leader PCR-based TB detection assay. In addition, the technique applies a disruptive force to the probe-target duplexes which makes detection highly specific even at room temperature. Thanks to its non- enzymatic detection approach, reagents and equipment are simple and robust, ideal for settings with limited resources. Our overall goal is to develop a molecular platform for TB testing that will be easy-to-use without training, accurate, rapid, inexpensive, portable and battery-operated. In this proposal, we will demonstrate all the properties of the final product by developing a non-automated TB assay and testing it using sputum samples spiked with known quantities of the bacillus. The focus of Aim 1 is to develop an efficient method to capture MTBC RNA targets from sputum samples containing MTBC cells. Aim 2 focuses on developing a multiplex SM-Scanning test and determining the limit of detection, cross-reactivity, sensitivity and specificity of the test. If successful, the new platfom could support other tests that are urgently needed in settings with limited resources such as TB drug susceptibility, Ebola and sexually transmitted diseases.